WE ISOLATION AND ANTIBIOTIC SCREENING OF TWENTYsEIGHT ACTINOMYCETES FROM AN ACID SOIL AND CULTURAL STUDIES ON SEVERAL SHOWING SPECIAL PROPERTIES ‘I’hom Io: the 0.9m OI M. S. MICHIGAN STATE COLLEGE EarI Jack Swarthout I954 IflESls . Thishtocertlfgthatthe thesis entitled The Isolation and Antibiotic Scremirg or Twenty-Eight Actinomcetea from an Acid Soil and Cultural Studies on Several Showing Special Properties. presented by Earl Jack Swar‘l'hou‘b has been accepted towards fulfillment of the requirements for Mgdegree mMfllogy flfw [Major proleasofl mamma— 0-169 ‘/ THE ISOLATION AND ANTIBIOTIC SCREENING OF TV‘I'ENTY— EIGHT ACTINOMYCEI‘ES FROM AN ACID SOIL AND CULTURAL STUDIES ON SEVERAL SHOWING SPECIAL PhDPERI‘IES by Earl Jack Syarthout A THESIS Submitted to the School of Graduate Studies of Michigan _State College of Agriculture and Applied Science in partial fulfillment of the requirements ‘ for the degree of IIDISTER OF SCIENCE Department of Bacteriology and Public Health Year 195A 'ffHESI-a ACKNOI’JL EDG EM INT The author expresses his appreciation and extends his thanks to Dr. H. J. Stafseth for his help in preparing this thesis; to Dr. E. H. Lucas for sup- plying the soil from.which the actinomycetes were isolated; and to Miss Lisa Neu and Miss D6213 Miller for supplying the bacterial cultures used in screening the actinomycetes. EOJOS. ii “3",? I” ‘2 "LJW' ';)')_ TABLE OF CONTHQTS Page HISTO RI CM BJkCKG EDUIID o o o o o o o o o o o o o o o o o l ANflnYSIS OF PEBBLE-{I o o o o o o o e o o o o o o o o e o ISOLATION OF ACTINOMYCETES FROM SOIL mPerj-mental O O C O O O O O O 0 I O O O O O O O O 13 Results and Discussion . . . . . . . . . . . . . . 20 SCREENING ACTINOMYCETIS FOR.ANTIBIOTIC ACTIVITY . . . . 22 Experimental 0 O O O O O O O O O O O O 0 O O O O O 23 Ream-ts and DiscuSSion O O O O O O 0 O O 0 O O O O 28 MORPHOLOGICAL AND CULTURAL STUDIES flpermental O O O O O O O 0 O O O O O O O O O O 0 #0 Results and Discussion . . . . . . . . . . . . . . #3 SWMMcooeoeoooooooooooooooooeé? LITERATURECITED....................69 iii LIST OF TABLES Table Page I. Formulae of the Eleven Media Tested in the Plate Dilution Culture Method for Isolating Soil Actinomycetes . . . . . . . . . . . . . . 16 II. Formulae of the Four Media Used in Screening the Actinomycete Cultures for Antibiotic Activity . . . . . . . . . . . . . . . . . . . 25 III. General Characteristics of the Twelve Test Bacteria Used in the Cross Streak Method for Demonstrating Antibiotic Activity of the Actinomycete Cultures . . . . . . . . . . . . . 29 IV. Inhibition of Growth on Four Media of Twelve Bacterial Species by Twenty-eight Actinomycete cultures 0 O O O O O O O 0 O O O O O O O O O O 30 V. The Twenty-eight Actinomycete Cultures Grouped According to the Range of their Antagonistic ACtIOD. o e e o e o e o e o o o e o o o o e e o 39 VI. Description of Organism 218 . . . . . . . . . . . #5 VII. Description of Organism 233 . . . . . . . . . . . 49 VIII. Description of Organism 2&1 . . . . . . . . . . . 53 IX. Description of Organism 58 and A15 . . . . . . . 56 X. Description of Organism 200, 201, 202, and 216 . 60 iv BISTO RICAL saw/Lemme The actinomycetes are a group of unicellular branch- ing microorganisms found widely distributed in nature, and seeming to form a transition group between bacteria and fungi, possessing characteristics related to each. The term ”actinomycete" refers to any member of the five genera Nocardia, Mycobacterium, Actinomyces, Streptomyces, or Micromonospora_ which comprise the order Aptinqmygetalgg. (l) Waksman (2, p. 22) gives the following characteristics for the order: Organisms forming elongated, usually filamentous cells, with definite tendency to branching; hyphae not exceeding 1.5 micron in diameter, .mostly about 1.micron or less. Usually produc- ing a characteristic branched myceliumm Mul- tiply by means of special spores, as well as by oidiospores or by conidia. The Special spores are formed by fragmentation of the plasma with- in the spore-bearing hyphae, the latter being straight or spiral-shaped. The oidiospores are formed by segmentation, or by simple division of hyphae by means of transverse walls, in a manner similar to the formation of oidia among the true fungi. The conidia are produced singly, at the end of special, simple or branching conidio- phores. They grow readily on artificial media and form.well developed colonies. The surface of the colony may become covered with aeria1.my- celium. Some of the organisms are colorless or white, whereas others form a variety of pig- .ments. They are either saprophytic or parasitic. In relation to temperature, most are mesophilic, though some are thermophilic. Certain forms are capable of growing at low oxygen tension. The latter description may also be found in Bergey's Manual 9;,Determinative Bacteriology. (3) The genera which are of interest antibiotically speak- ing are Nocardia, Micromonospora, and Streptomyces. Of these the most interesting and extensively studied is the genus Streptomyoes. (A) In this genus are placed the aerobic nonpathogenic aotinomyoetes producing aerial mycelium and multiplying by forming true conidia in chains. (3) Cohn (5) in 1875, was the first person to study an actinomycete, under the generic name of Streptothrix. From that date through 1943 over 31 different generic names were used in designating all the actinomycetes or certain constituent groups. (2) The antagonistic action of actinomycetes to the growth of other.microorganisms was first demonstrated by GaSper- ini in 1890. The actinomycete, apparently the same as Cohn‘s was able to live on the surface of bacteria and fungi because of its ability to digest the membrane or cell wall of the lower fungi. (2) One year earlier the word "antibiosis" was coined by Vuillemin in reference to antagonism between two living forms. ‘Ward (6) ten years later, applied the word in re- spect to microbial antagonism and it has lasted to the present time. The theory of antibiosis really obtained its start, however, in 1917, when Greig-Smith (1) observed the inhibi- tion of certain spreading colonies of Bacillus mycoides and Bacillus vulgaris on nutritive agar by soil actino- mycetes. In 1921, Lieske (2) tested a large number of actino- mycetes for their antibacterial effect and succeeded in showing that certain bacteria pathogenic to man may be in- hibited by some excretion of the actinomycete which he be- lieved to be a Specific bacteriolytic enzyme. Three years later, a "Streptothrix" was isolated from the air by Gratia and Bath (6) which lysed dead or living staphylococci. From 1936 to 19h1, welsch (6) did extensive work on an organism very similar to the one described by Gratia and bath. The bacteriolytic substance was named Actinomyoetin and the organism, Streptomyces alpgg G. Rosenthal (2) obtained an actinomycete culture from dust which inhibited living Clgstridium diphtherium and lysed the dead cells. At the same time (1925) he intro- duced suitable methods for measuring the bacteriostatic and bacteriolytic properties of actinomycete cultures. At this time other researchers became interested in the antagonistic actions of the actinomycetes as a group and so large numbers of actinomycetes were being worked with instead of only one or two cultures. The first detailed study on the distribution of an- tagonistic actinomycetes was done in 1937 by Nakhimovskaia (A). She reported that 58.75% of 80 actinomycete cultures isolated from a variety of soils possessed antagonistic properties. A year later Krassilnikov and.noreniako (2) reported that species primarily in the genus Streptomyces were an- tagonistic to a variety of organisms particularly the gen- era Nocardia, gycobacterium, and Micrococcus. The activity was believed to be due to a single substance possessed by all the species tested which were active. The substance studied by these and other Russian workers was thought to be similar to lysozyme, a 1ytic enzyme. Kriss (l), in 19A0, was one of the first workers to try to isolate the antagonistic substance using various sol- vents, but he had little success. By 1942 researchers through- out the world were beginning to organize the previous works. It was concluded that actinomycetes possessing antagonistic properties are widely distributed in nature. waksman (7) found 43.h% of 2th cultures isolated at random from a vari- ety of soils possessed antagonistic properties. Alexopoulos (8) found that 56.25% of 80 cultures were antagonistic to a fungus Colletrotrichgg,glggsporioideg. Out of 660 cultures of actinomycetes isolated on a nonselective basis from soils from 5 locations in northern Canada, 61.2% showed antagonism against at least one test organism out of the eight that were tested (9). Because of the widespread and develOping interest in these antagonistic substances, it inevitably followed that a substance would be found which would be of practical clinical use. This occurred in 19th when Waksman (10) published his discovery of Streptomycin after examing some thousands of actinomycetes, hundreds of fungi, and many bacteria during the preceding five years. With this discovery and the drug's subsequent use in .medicine to kill pathogenic gram negative bacteria igflligg, the subject of.microbial antagonism took on its greatest significance. It was at this time that the word antibio- tic came into widesPread existence. Waksman (11) defined the word "antibiotic" as a "substance produced by micro- organisms which has the capacity of inhibiting the growth or even destroying other.microorganisms." A slightly different definition was suggested by Benedict and Langlyke (12). They defined it as a "chemical compound derived from or produced by living organisms which is capable in small concentration, of inhibiting the life processes of.microorganisms." Immediately following the discovery of Streptomycin, the pharmaceutical industries began their own research on the actinomycetes and in antibiotic screening. Large plants were constructed for the production of Streptomycin and for large scale soil investigations. Antibiotics were sought 'which were of low toxicity and yet had a wide spectrum of antibiotic activity, that is, they were actively antagonistic to a number of.microorganisms composed of different physi- ological types such as gram positive and gram.negative bac- teria, fungi, etc., and yet were not poisonous to animals. Streptomycin is a wide spectrum antibiotic primarily active against gram negative and gram positive bacteria. The work of isolating antibiotic producing actinomycetes from various soils gathered throughout the world was begun in earnest with the discovery of this antibiotic, and is still being carried on. Industrial laboratories are organized for.mass screen- ing programs and search thousands of soil samples yearly. The number of actinomycetes studied is small in relation to the number isolated. The industries are interested only in the antibiotic which will compete favorably with those already on the market. Antibiotics that have entered the production stage are few compared with the large numbers which have been found. The work reported in this paper, and other research like it, is justified by the very fact that the industries are organized for mass screening only. Their time spent on any particular soil sample and the organisms isolated from it is restricted. They are not interested in the number of active cultures found, or in the classification of the actinomycetes whose antibiotics will not compete favorably with those on the market today. Although the work in this thesis is restricted to a single soil, it is spent on a more thorough investigation of the cultures isolated from it. The purpose of this work was to obtain from a soil as many actinomycetes as possible showing either antagonism.toward a wide Spectrum of bacterial species or showing other special properties. The cultural and morphological characteristics of these were investiga- ted and the cultures were identified if possible. ANALYSIS OF THE PROBLEM When a problem is selected in which the particular group of microorganisms to be investigated is a part of a mixed microbial population, the problem automatically enlarges itself to include the isolation of that particu- lar group. In this work, the actinomycetes existing in a par- ticular soil sample were wanted for subsequent checking of their antibiotic properties, and the classification of those showing special and/or unusual properties. Several methods have been used for isolating antibiotic organisms from soils and other sources where mixed.microbial populations are found. All the methods, however, are vari- ations of one called the "crowded plate technique" which in turn is only a Special case of the "plate dilution cul- ture method". Instead of isolating all colonies exhibiting similar morphological characteristics as in the latter method, only colonies exhibiting zones of inhibition are selected in the "crowded plate technique". The.major disadvantage to this method is the uncer- tainty as to whether an appropriate test organism exists in the soil which will Show up the antibiotic activity of all the organisms in the group being investigated. A summary of the techniques for isolating antibiotic organisms from.mixed cultures is given by Flory, Chain, and coworkers in Antibiotics. (v.1, pp. 81-88) For the evaluation of the actinomycete population of a soil and the isolation of cultures for later antibiotics work, the plate dilution culture method is used most ex- tensively. (l),(2). In its simplest form, this method consists of plating out a suspension of soil with a suitable medium, incubating, and then selecting the particular type of.microorganisms sought for future investigation. Before the method can be used satisfactorily, however, certain factors such as the type of nutritive medium and its reaction, the temperature and time of incubation, and the treatment of the soil prior to incubation.must be decided upon. To date, there has been published no data comparing the different factors and cfonditions used by past investigators. Personal choice has been the governing factor of such a selection. (A) For this reason, it was thought advisable to include the isolation as the first part of the exPerimental work of this problem. Results were obtained by varying the dif- ferent conditions and attempting to select finally a park ticular medium, reaction, incubation temperature, and soil treatment which would give results comparable to those obtained by using several variations of each factor used in the plate dilution culture method. 10 After isolation, the actinomycete cultures were screened for antibiotic activity using the cross streak method. Since the beginning of the search for antibiotics, many methods and special techniques have been developed for de- termining not only whether an antibiotic is produced by a given organism, but also the quantity produced. A very good summary of these methods may be found in Antibiotics. (v.1, pp. 75-81) There is one method which has been important from the start and is still the most commonly used for testing not only for an antibiotic, but also for the number and types of organisms upon which the antibiotic acts. This method is the " cross streak method" developed by Garré (l) in 1887, and is now widely used in industry as a primary screen- ing method where many actinomycetes are to be checked for antibiotic properties against many other microbes. The procedure is as follows: The antagonist, i.e., the actinomycete is streaked across a nutritive agar plate which is then incubated at a suitable temperature until the organism has grown out abundantly. Then secondary streaks of the test organisms are.made at right angles to the ac- tinomycete streak. The plate is again incubated, this time at a temperature optimal for growth of the test or— ganisms. If the actinomycete has produced an antibiotic antagonistic to a test organism, then the latter will be inhibited in the neighborhood of the actinomycete growth at 11 a distance proportional to the sensitivity of the test organism to the antibiotic, and the amount and diffusibility of the antibiotic produced. The type of medium used to produce antibiotic activity and the types of test organisms used to detect this ac- tivity are discussed later in relation to the procedure of screening the isolated cultures. After all the isolated actinomycete cultures were screened for antibiotic activity, several Showed certain properties that made them worthy of further investigation. Classification of these cultures was therefore at- tempted. There was the possibility that either a previously unknown organism, or a known organism with uncharacterized properties would be found. The work of classifying ac- tinomycete cultures, however, is complicated by the fact that there has been found no morphological or physiologi- cal characteristics which.may be used to divide the species of a particular genus into smaller definite groups. (A) There are 73 species of the genus Streptomyces listed in Bergeyfs‘Manual g; Determinative Bacteriology. (3) There has been found to date no natural breakdown of this number of species into smaller groups. A grouping based on the color of pigmmts on certain types of media, and morphology of the species has been attempted, but due to the vari- ability of composition of these media, the variability of pigment production, and the use of no specified medium 12 upon which the morphology was studied, the grouping is slightly better than none at all. It can be seen then, that when the classification of an actinomycete culture is attempted, all the known species of actinomycetes.must be checked individually against the unknown culture. Waksman (5) published a compilation of the character- istics of 41 different species of soil actinomycetes in 1919. The descriptions included the morphological, cultural, and biochemical characteristics of each. These descriptions may also be found in Bergey's Manual. Due to the extent of Waksman's work, some of the.media which he used were prepared for this work so that the cul- tures worked on in this paper could be compared with those of his. Other.media used in Bergey's Manug1_were also prepared. - The results of isolation, antibiotic screening, and classification are summarized on page 67. ISOLATION OF ACTINOMYCETIS FROM SOIL Experimental The plate dilution culture method was decided upon for isolating the actinomycetes from the soil. All other .methods used for isolation of these organisms are special variations of this.method and limit the worker to the iso- lation of only the actinomycetes which Show antibiotic activity in the isolation medium. As stated on page 9, the plate dilution culture method consists of plating out a suspension of soil with a suitable medium, incubating at a given temperature, and selecting the particular type of.microorganisms sought for future work. To date, there is no set of conditions universally accepted for use in isolating actinomycetes from soils. The reason for this may be that the different soils inves- tigated vary broadly in their organic contents and reactions, thus also varying their.microbia1 population. The pH of the soil sample used in this work checked at 5.1 to 5.3. This reaction is very favorable to the growth of fungi but not to actinomycetes. It would be difficult merely to select a particular.medium and the various other factors which would allow the actinomycetes to produce good growth while restricting the fungal growth. The first step then was to find a nutritive medium which 13 14 would not allow fungi or bacteria to grow in such profusion as to make the isolation of the slower growing actinomycetes impossible. If possible, no surface reductants or fungal inhibi- tors were wanted in the procedure. There is a possibility that they inhibit actinomycetes as well. Many media have been used in the past for isolating actinomycetes from soils. The following media were tested here: 1. actinomyces agar (13) 7. .malt agar 2. Czapek's agar (l4) , 8. North gelatin agar (17) 3. Jensen's agar (15) 9. nutrient agar (2) 4. nitrate sucrose agar (2)(16) 10. potato dextrose agar 5. streptomyces agar (1) 11. S-l agar 6. brain-heart infusion agar (17) The composition of each medium and its reaction can be found in table I. Twelve plates of each medium were inoculated with soil diluted from 102 to 10", four plates per dilution. The plates were incubated at room temperature and observed for actinomycete growth every day until they became over- grown with fungi. On the third day, 8 cultures were iso- lated. One was from actinomyces agar, 2 were from strep- tomyces agar, and 5 were from nitrate sucrose agar. The colonies on the nitrate sucrose agar were all between 2 and 3.mm. in diameter, while the other 3 colonies were only about 1 mm. in diameter. All were isolated from plates of the lowest dilution. 15 After only four days incubation, it became evident that most of the media tested were inadequate for use with this particular soil. All three dilutions of media numbers 3, 5, 9, and 10 were covered by Spreading growths of both bacteria and fungi. Media numbers 2, 6, 8, and 11 had Spreading type bacteria dominating the plates. All the others except number 4, nitrate sucrose agar, had fungi covering the plates. On the plates of nitrate sucrose agar, the bacteria and fungi grew out in well-defined colony formations which allowed time and room for actino- mycete growth to take place. however, only three more cul- tures were obtained. The three cultures obtained from the actinomyces agar and streptomyces agar were cultivated in sterile plates of the same medium from which they were isolated, then trans- ferred onto nitrate sucrose agar plates to check for growth on that medium. All three were found to grow out abundantly. For this reason, plus the fact that nitrate sucrose agar had the least interfering growth, it alone was chosen for subsequent isolation work. At this time, the method of inoculation was changed. The soil which had been diluted was now added directly to the surface of the hardened agar plates and spread by means of a sterile needle. In this way it was hoped that more actinomycete growth would be evidenced. 16 TABLE I Formulae of the Eleven Media Tested in the Plate Dilution Culture hethod for Isolating Soil Actinomycetes Culture Media redients grams/liter ar rain heart infusion ar t ar otato dextrose ar ctinomyces ensen's itrate sucrose orth Gelatin treptomyces utrient r 6 one one se n e n us on e infusion ota infusion e ex 0 ' umen ex rac ucrose ex rose a ce um as a um citrate 17 It has been noted (18) that the spores of the actino- mycetes have waxy coatings which are not readily wet. They will therefore float on the surface of a liquid. Thus many of the spores will be missed when obtaining a dilution sample using a pipette placed beneath the surface of the dilution liquid. After the selection of nitrate sucrose agar as the isolation medium, its pH was adjusted from.6.0 to 8.0 in steps of 0.5. Colonies of actinomycetes were found only on the plates containing the medium at a pH of 6.5 and 7.0. Allen set the pH of nitrate sucrose agar at 7.0. The medium after autoclaving and without adjusting was found to be 6.8 t .2. No noticeable results were found between the unadjusted agar medium and that adjusted at 6.5 or 7.0, and so the unadjusted nitrate sucrose agar was used. It should be noted that cultures were isolated whenever possible. Soil inoculated plates of nitrate sucrose agar were incubated at three temperatures - room temperature (approxi- mately 2500), 35° 1 1°c, and 54° 1 2°C. Waksman (5) found that most of the actinomycetes will grow readily at temperatures ranging from 15°C to 37°C, the higher temperature favoring a more rapid growth. The tem- perature of 54° was included to grow out any thermophilic Species from the soil. 18 A few cultures were isolated at room temperature which would not originally grow at 35°C. On subsequent attempts the cultures began to produce slight growth at the raised temperature, but they would have been missed if only the higher temperature had been used for isolation. Only one species was isolated at 540 I 2°C. The majority of the cultures were obtained at 35° 1 1°C. Thereafter, the following conditions were set forth in the procedure for the cultivation and isolation of the soil actinomycetes. Plates of nitrate sucrose agar were poured approximately 20 m1. of agar medium per plate, and allowed to harden. The soil which was kept refrigerated at 4°C was inoculated onto the surface of the agar in a quantity of .15 - .2 grams per plate. Twigs and other debris within the soil were included with the soil. The plates were then incubated at three temperatures — room temperature, 35° 1 1°C, and 54° t 2°C. The plates were checked after 3 days incubation and every day thereafter for about a week. Suspected actino- mycete colonies were picked from the plates by means of a sterile inoculating needle and transferred into a sterile nitrate sucrose plate for primary pure cultivation. Cultural characteristics which identify actinomyces colonies are the following. The agar colonies are usually small and tough, adhering to the.medium. They are either flat or semicircular and growing deep into the medium. If 19 they are semicircular, they may exhibit radial folds on the surface, or they may be smooth and shiny. If they are smooth and shiny, they are distinguished from bacteria in that the colony is leathery and can not be broken up. It is removable all at one time. Almost all actinomycete growth gives off a typical earthy odor when cultured which readily identifies them. IMicroscopically, actinomycetes .may be distinguished from.other organisms primarily by the production of branching mycelium not exceeding 1.5 microns in diameter and usually only about 1 micron in diameter. All actinomycetes are gram positive. Cultures were isolated and kept on the basis of their diverse appearance. Presumably, one could isolate cultures as long as the sampling material held out. However, there is, to date, no published information dealing with the iso- lation of actinomycetes on any basis other than random sampling or diversity of appearance. Cultural character- istics of the isolated cultures were checked against each other on nitrate sucrose agar and S-l agar. Dif- ferences in the rate of growth, contour of the surfaces of the colonies, edge of the colonies, color of the vegeta- tive mycelium, color of the aerial mycelium, color of the spores, or color of any soluble pigment were searched for. If any of the isolated cultures differed from the others in any way mentioned it was placed on an agar slant of nitrate sucrose agar and one of S-l agar, incubated for 20 three days, and then refrigerated until it was to be tested with the rest for antibiotic activity. Results and Discussion Twenty-eight cultures were selected for screening of their antibiotic activities. Records were not kept of the actual number of colonies isolated from the soil inoculated plates. The isolations were continued until no further cultures were isolated which differed from those already obtained. This method seemed justified in two respects. First, during the final isolations, no different appearing cultures were found. Second, in random sampling it would be possible to pick many colonies of the more profusely growing strains of actinomycetes and yet miss cultures which appeared rarely on the plates. This would later involve the screening of many identical cultures. If the non-antibiotic strains of actinomycetes were the commonest in the soil, then the work would be of no value. It is interesting to nOte that on the soil inoculated plates which were incubated at 54°C, only one actinomycete was found. This actinomycete showed rather peculiar prop- erties. It produced abundantly a deep, bluish-purple water soluble pigment that when treated with hydrochloric acid turned bright red. The culture also gave off a pleasantly smelling fruity odor instead of the usual musty or earthy odor so often associated with actinomycete growths. 21 0f the other 27 cultures, only 3 showed any water soluble pigments. However, most of them produced pigments within their mycelia or spores. When checked morphologically, some cultures produced spiral formation in their aerial mycelia while others produced no aerial mycelia. Some cultures produced conidia readily while others produced none at all. Because of the large number of variations in charac- teristics shown in the 28 cultures, it was expected that there would also be shown many characteristic Spectra of antibiotic activity. SCREENING ACTINOMYCITES FOR ANTIBIOTIC ACTIVITY A major difficulty which confronts any worker in the field of antibiotics is that of selecting conditions which will give favorable results. Failure to use the proper medium when testing for antibiotic activity can give negative results with organisms which actually are good antibiotic producers. This fact is illustrated by several industries which have included in their patents the formulae of the culture media used in producing their antibiotics (2)(19). The selection of the temperature and time of incubation is also just as important as the selection of the medium. Organisms vary considerably in these respects. The optimal temperature for antibiotic production is not necessarily the cptimal temperature for growth. And antibiotic pro- duction by a particular organism may reach its maximum af- ter a few days incubation, or after a few weeks. Last, but not less important, is the critical selec- tion of the test organisms. Unless an antibiotic is sought for its action against a specific organism, a wide variety are used in testing for antibiotic activity. Streptomycin would hav:e been missed if tested against only yeasts or fungi. Penicillin, which is very active against gram positive bacteria would not have been discovered if tested against gram negative bacteria. 22 23 The preceding paragraphs were included in order that the reader could understand more clearly the problems which had to be met before selecting the conditions used in this work for screening the isolated cultures. Ekperimental Nutritive agar plates were poured two days before using to check for sterility and to allow the surface of the agar to dry. The antagonist, 1.6. the actinomycete, was streaked across one Side of the agar plates by means of a loop in a path 5 mm. in width and about 25 mm. from the edge of the agar at the middle of the streak's length. The plates were then incubated at 35° 1 1°C for about 60 hours. At that time, secondary streaks of the test bac- teria were made at right angles to the actinomycete streak. The secondary streaks were.made by means of a loop, streaking away from the actinomycete. Twenty-four hour broth cultures of the bacteria were used. After the secondary streaks were made, the plates were again incubated at 3500. This temperature was close to the optimum temperature for all the test organisms except Sarcina 13333. Its optimum temperature is given in Bergey's Manual as 25°C. Hewever, an excellent growth was obtained at 35°C. After another 12 hours, the plates were removed from the incubator and readings were made in millimeters of the 24 zone between the actinomycete streak and the bacterial growth. Readings were made by placing a celluloid ruler on the bottom of the Petri dish with the scale measurement superimposed over the streak of the test organism. A small microscope light held under the inverted culture plate gave a well defined end point to the test streak. Readings were made only to the nearest millimeter. Greater accuracy was not needed. The measurements were taken for purposes of comparing the cultures with one another and for comparing their inhibitive effects on the different physiological types of test organisms such as the gram positive and gram negative bacteria. The results are recorded in table IV. The media which have been used for the production of antibiotics run the gamut of nutritional substances. Many of them contain tryptone plus some particular carbohydrate. Others contain meat extracts and peptones, with or without sugar added. With this in mind, four media were prepared for this work which contained some of the common sources of nitrogen and carbon usedflin the past. The media were also selected so that they would supplement each other to a certain extent. It was thought that perhaps in this way, the production of an antibiotic may be found to be connec- ted with the use of a certain sugar or other substance. The formulae of the four media used for screening the actino- mycetes are given in table II. 25 1w, ha. I . news mm.~ - . usommmm w m - - - .aer m cm: nomopmhna m N n n cmonosm w 54 u u n no mm.~ - . sommmg m m - - . Hoez m cm: ncmopmhna m m u n omoosao 7m.Nmu u u u uncm¢JIMIIN4HI a a m can a n adepmmm w m u - n . -Hoez w m upomnpwo Hoom m can a a cmoosac HSWfiI w o u n a sodopmom m m u pomnpuo some» Mm.a u pownpwo Homm Howw omonodm omoumhna Hmm¢ opoflmofimr omothhB . Hows omoosamrw Hom¢ omen hmmmwmom Assesses oflpoanssaa you monepaso opoosaoeasos we» wowqoohcm SH comb caves 990% on» no ooadsuom HHHQAM4B 26 The screening of each culture was carried out on four .media because it was known that an antibiotic may be pro- duced in one medium.and not in others. Possibly it would be worth while to run the tests on several media of vary- ing composition. Whatever the.medium used, however, it .must not only allow the actinomycete cultures to grow out, but, just as important, the medium must allow the test organisms also to produce growth. These four media met the conditions very favorably. The reactions of the.media were unadjusted. The major reason for this was the fact that after checking the reac- tions used for 16 media employed in the industrial production of actinomycete antibiotics such as Chloromycetin, Strep- tomycin, Aureomycin, and Tyrothricin, it was found that they varied from 6.0 to 7.2 before inoculation. After incubation the variance was from 4.5 to 9.0. These vari- ations occurred not only between different cultures, but also for the same culture using different media. Variations could also be found in the reaction of a single medium a depending upon the number of days incubation, and seemingly unaffected within certain limits by the original reaction of the medium. (2)(19) There appeared to be no evidence which would support the adjustment of the reactions already shown by the four media used in this work. All actinomycete cultures produced luxurious growth after 60 hours incubation at 35°C. Sporulation.also began at this time. 27 If an antibiotic were produced by any culture, it should at least have been produced in a colony containing all stages in the actinomycete's life cycle. It was therefore concluded that the test organisms could be streaked at this time and any antibiotic cul- tures would be demonstrated. Twelve hours of incubation at 35°C was sufficient to produce heavy growth streaks of the test bacteria and so readings were taken at that time. The total time elapsing between streaking the actinomycetes and reading the plates for antibiotic activity was 72 hours. The streaks of the test organisms were.made from brain heart infusion broth cultures incubated at 350C. Twelve bacterial Species were selected for testing the antibiotic activity of the actinomycete cultures. It is necessary during the isolation of antagonistic organisms and the study of the antibiotic substances to use more than one test organism including one or.more gram positive and one or.more gram negative bacteria. Antibiotics vary greatly in their action on different organisms possessing different physiological prOperties. The antagonistic action of a given antibiotic may be highly selective, affecting detri- mentally only a single strain of a bacterial species, or it may act generally, not only on a given species but also on gram positive and gram negative bacteria and fungi as well. In most methods of testing for antibiotics, the choice 28 of the test organisms rests with the experimenter. There is no standard procedure developed through rational plan- ning for selecting them. Most of the general groups of organisms found in any bacteriology laboratory have been used for this work. In general, the only factor to be considered in selecting the test organisms is that they are picked from a variety of physiological groups. For this reason, although the work was limited to bacteria, both gram positive and gram negative bacteria were used. In these two groups, both pathogenic and soil types can be found. In the gram positive group, both rods and cocci are present. See table III. Results and Discussion Table IV shows the antagonistic properties of 28 actinomycete cultures tested on each of four media against 12 bacterial Species. I The actinomycete cultures producing wide spectra of antibiotic activity are seen to be organisms 241, 58, 201, 216, 415, 200, and 202. Organisms 58 and 415 produced their antibiotics on all four media, although decreased activity was shown on penassay base agar by number 58. Apparently, they are able to utilize a number of carbon and nitrogen sources for synthesizing their antibiotics. Organisms 200 and 202 were antagonistic to the test organisms only on penassay base agar. This may mean that a more complex 29 oHSpHdo caojdoadmwompmm, GOA mHHuQS mIoH mmodflwohom mmdoaoSSomm owphomonmom con maflpoa I manom45> mooponm _amflqmwao Haom mdoooo mu cocoa enaonow oaaowoopmm. moon I Hoodoo mHHomHnm oHHpoaIaod canomoopom, mSoooo x sum mooooOOpmoapm copwaoma moon I sonoaadm oaaodoaaom sawmmnml.oaqmwoopwo mammoaIaod I ‘ uopmaoma much oHHpoa. I wmoqfimonom mmdoaouoomm manhohm..ofidowomemm III cadomoopmm, moon I omaqoaooqmwwadoflmnodm .cmpeasmmeodo oHHmoSIooq ommomfic quaumopsfi aomw. 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O J 0/99 1 n9 0U. .01. B 98 U 0 HE .A KT. .JS m0 1.: HT: .1 mm. 18 89 To 0.0 Gm. OO ztnun can+ and Had a TI nnu m2! tiq 11L U. 9“; can. 98 BI. 99 d 00 SD. 8 a IT. 00 90 IO rtu U TI Q IU To OI. TSSIL SU U0 . um IS 9 T. O a UW U9 I. In 99 90 00 s e o I oo ILL 0 ss I so mopoohaoSHpoe. Bu 0 I SU BI q I O n.% w a emw 9.5 m. e m SSS m SHUSS SMHSSwwo Smear. coSSHpSoo .PH ”wage TABLEIIV continued #— Test’Organism €‘9I esourSnxee seuomOpnes spxeSInA sneeoxa saint euromes reduce attefitus °de sncooo ~oqdexis mnmottnd etteuomres esourSnJee seuomOpnes Ud 1293 II00 etqorxeqosg sarcomas ettersqe Stttnqns SHIIIOBS esoqdfii attsuomTes seoeSch snoooooxorw Media and Actinomycetes I I ORGANISM 214 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 tryptose phosphate tryptose sucrose glucose agar ,penassay base ORGANISM 213 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 tryptose phosphate tryptose sucrose glucose agar ,penassay base ORGANISM 217 33 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 glucose agar tryptose phosphate tryptose sucrose penassay base ORGANISM 223 0000 MOON 0050 0000 0000 0000 0000 0000 I~t\00’~f\ 0000 0000 glucose agar tryptose phosphate tryptose sucrose ,penassgy base ORGANISM 233 000I-I r... 0000 0000 H 0000 0000 0000 0000 0000 000r-I ,_.' 0000 0000 0000 glucose agar tryptose phosphate tryptose sucrose penassay base Inhibition in millimeters demonstrated by cross streak method 34 doSWSS MSoHMm mmono ho copprmSoaow mHmpoSHHHHS Sm SOHPHSHSSH 0000 0000 0000 0000 0000 {-9I esourSnJee seuomOpnesg LAOOO 0040 0000 0000 0000 stxeSInA snsqoma SH mH 4H NH (“0011) 0000 0000 H saint euromes \0000 0000 0000 0000 0000 pounce Btrefitqs MONO moon 0000 \OH‘IMN \Ol‘l‘fl) Nr-I °ds snoooooqdemas 0000 0000 0000 0000 0000 mnxottnd eIIeuomTes mH 4H 0H NH 4H 0H mH w 0:: 0005 0000 0000 0000 0000 0000 0000 4- H 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 m r-l ITOO esourSnxse seuomOpnesg eeruomneud BTIBISQSIX erqorrsqosg SIIIQQHS SHIIIOBS 0000 0000 0000 0000 0000 esoqdfq etteuomxes mH 5H 0H 4H 0N SH SH 0H neoH 0000 0000 rdri r4 seueSofid snooocomotw ommn1wmmmmSoq omonoSm omopohnp oprSmoSS omopmhnp Home omooSHw mHN_Emszumo omen mommmSQm. omonoSm omopmsnp opmnmmoam omopmhnp Home mmooSHw 4H: EmHzmumo omen NommmSom omoHoSm cmoumhap mpmSmmoom omopohnp Home cmooSHm mom smHzaamo omen NommSSvm. omoaosm cmOpmzap opmSSmoSm omOSSSHp Home omooSHm mmN‘SmHZSUmo omen MSmmSSoQ. omonosm omopmmnp opmSSmoSm omopmhnp Home omooSHw Hem SMszamo mopeohEOSHHSS SSS eHems SmHSSwno pmww USSSHpSoo >Hanm¢B TABLhIIV concluded Test OrganiSms E-9I esourfinasa ssuomOpnesa sIJBEInA snaqora ssqnt euroxss teuuos attefitus °ds snoooooquaqs 'anoIInd atteuomTes esourfinree sauomopnssg earuomneud PITBISQSTX ITOO epqorxeqosg SIIInqns snIIIoaa asoqdfiq atteuomtas ssuefiofid snooooozorm Medla and Actinomycetes WMOO H 0 WAN IDM 1+1 7 0000 0000 0000 0000 0000 0000 tryptose phosphate tryptose sucrose ppenassaprase glucose agar ORGANISM 421 0*— 35 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 glucose agar tryptose phosphate tryptose sucrose penassay base 0 IGJWISM 431 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 0000 glucose agar tryptose phosphate tryptose sucrose penassay base OhGANISMI27h 0000 0000 m00d) 0000 000m 0000 0000 0000 0000 #00m 0000 0000 Inhibitibn in millimeterswaemonstrat6&“b1 cross streak.method tryptose phosphate glucose agar tryptose sucrose penassay base 36 substance is needed such as the yeast extract before the antibiotic substances can be produced. A slight inhibi- tion of Bacillus subtilis on tryptose phOSphate agar was produced by both 201 and 216, however, because the activity is slight it is only mentioned in passing. Other than this slight inhibition, organisms 201 and 216 resemble organisms 200 and 202 in producing their antibiotics only on penassay base agar. Another organism.which produces its antibiotic only on penassay base agar is organism.233. Its spectrum of action seems to be against specific gram positive and gram negative bacteria. In the group acted upon, however, are found both soil organisms and pathogens. There is some doubt whether organism Zhl produces one antibiotic or two. This question arises upon noting that only gram positive bacteria are inhibited upon three of the four media, while on the fourth, glucose agar, in- creased activity against the gram positive organisms is found and at the same time inhibition of two gram.negative or- ganisms occurs. There are two possible eXplanations for this phenomenon. First, the greater activity shown on the glucose agar.may signify that the actinomycete has a fas- ter growth rate on this medium than on any of the others. It may be that a second antibiotic is produced later and has activity against gram negative bacteria only. Second, it may be that only one antibiotic is produced by organism 37 241 and due to greater production in glucose agar it exhibits inhibition against organisms which are antago- nized only by larger amounts of the antibiotic. In comparing the four media, penassay base agar gave the best results allowing 75% of the actinomycetes to pro- duce inhibition against at least one of the test bacteria. Tryptose phosphate agar, glucose agar, and tryptose sucrose agar allowed activity to be shown by 53.6%, 50%, and 46.4% of the actinomycetes in the order stated. Not only would a reduced number of active cultures have been observed by using a single medium, but also cer- tain activities exhibited on different media by specific cultures would have been missed. Organisms 223 and 274, for instance, inhibit just gram positive bacteria on penassay base agar and glucose agar. On tryptose sucrose agar, number 274 inhibits a gram negative organism and number 223 inhibits two gram negative organisms. This would seem to indicate the production by each of them of two different antibiotics. In addition to the group of organisms showing activity against a wide Spectrum of the test bacteria, three other active groups of cultures may be found. See table V. First, there is the group of 14 cultures which in- hibited Just gram positive bacteria. Second, there is a small group of only three cultures which produced activity on certain media against just gram negative bacteria. 38 Third, there is the group of cultures which produced an- tagonistic affects only upon specific bacterial species. Organism 203 inhibited only Bacillus subtilis on all four media. Organism 413 inhibited Sarcina lutea on penassay base agar and glucose agar. Organism 214 inhibited just Proteus vulgaris on tryptose phosphate agar. Some of the first two groups of organisms are also placed here. 39 TABLEIV The Twenty-eight Actinomycete Cultures Grouped According to the Range of Their Antagonistic Action Group 0 Inhibition against neither gram positive nor gram negative bacteria Penassay base agar - Glucose agar - Tryptose phosphate - Tryptose sucrose - Inhibition against gram positive 419, 214, 218, 421, 37, 217, 305 201, 216, 419, 37, 200, 202, 204, 214 218. 217, 233. 305. 421. 431. 413, 37, 200, 202, 204, 218, 217, 273, 274, 305, 421. 431, 233. 201, 216, 413, 419, 37, 200, 202, 431, 204, 214, 213, 217, 233, 274, 305, 421. Group I bacteria only Penassay base agar - Glucose agar Tryptose phosphate Tryptose sucrose 203, 241, 203, 425, 201, 414, 203, 254, 273. 254, 273, 203, 215, 417. 408, 414, 413. 274. 216, 417. 254, 413, 215, 408, 254, 241, 425, 37, 204. 223, 417, 223. 408, 408, 274. 414, 215. 417. 419, 425. 241, 425. 414, 215. Inhibition Penassay base agar Glucose agar Tryptose phosphate Tryptose sucrose Inhibition Penassay base agar Glucose agar Tryptose phosphate Tryptose sucrose against gram negative Group II bacteria only none {1011 e 223, 214. 223, 273. Group III of only 1 or 2 bacterial 203. 413, 37. 204. 273- 203, 254, 413, 273, 274. 203, 216. 254, 419, 214, 203, 254, 223, 241, 273, Group IV species 223.1d7 417 Inhibition of both gram positive and gram negative bacteria Penassay base agar Glucose agar Tyyptose phosphate Tryptose sucrose 58, 201, 58, 415, 58, 415. 58. 415. 200, 202, 216, 415, 233. 241, MORPHOLOGICAL AND CULTURAL STUDIES EXperimental Nine cultures were selected for further study on the basis of antibiotic activity or other properties shown by them in the course of this work. Organisms 58, 200, 201, 202, 216, and 415 each pro- duced inhibition of a wide spectrum of bacteria. Organism 233 showed activity primarily against gram negative bacteria although it also inhibited Sarcina lgtgg, a gram positive coccus. It was also peculiar in its antibiotic production on only one of the four media upon which it was tested. Organism 241 was unusual not only in its production of principally a gram positive antibiotic, but also in its production of a litmus-like pigment and its optimal growth temperature of 54°C. It was also the only organism iso- lated which gave a sweet, fruity odor instead of the usual earthy odor associated with actinomycetes. Organism 218 was the only culture isolated that produced a brown, water soluble pigment which was observed in almost every protein containing medium in which it was grown. It was also one of the five cultures which produced no activity whatsoever. Classification of it was attempted merely because it was distinct from the other 27 cultures by its pigment pro- duction. 40 41 In the past, identification of antibiotic producing actinomycetes has been attempted on the basis of morphology, pigment production, and/or physiological characteristics such as nitrate reduction, starch hydrolysis, and ability to utilize carbohydrates. All have been found to be rather unsatisfactory procedures. Henrici (20) stated that it was not possible to iden- tify most of the species of the genus Streptomyces which he had isolated. He believed that as much could be said for abandoning the species concept for this group as for any other group of microorganisms. Routein and Finley (4) believed that any taxonomical work on this genus could be only provisional with reference to given cultures deposited in culture collections. Benedict (21) said that he was forced to conclude "that the present system of classification of the actino- mycetes, which is based on morphology and relatively few physiological tests, especially for the genus Streptomyces, made it extremely difficult for competent investigators to 'key down' an unknown culture." ' Although it's quite possible that no natural relation- ships have been found in the group as yet, there has been a good deal of work done on the individual species. These Species are listed in Bergey's Manual and are separated from each other mainly by cultural differences, i.e., their growth on various media. 42 In this thesis, classification was undertaken by preparing the various media mentioned in Waksman‘s clas— sification work of 1916 (17) and 1919 (5) which accounts for over half of the species descriptions found in Bergey's Manual for the genus Streptomyces. The other media pre- pared were selected from Bergeyfs Manual. On the solid media prepared, the cultures were ob- served for type of vegetative growth, presence and color of aerial mycelia, and the presence and color of soluble pigments. The cultures grown in liquid media were observed for type of growth, presence of soluble pigments, and special reactions due to the particular type of medium. Morphologically, the growths on certain solid media were observed microscOpically for straight or spiraling conidiophores and the presence of Spores and their shape if present. Two procedures were followed in checking the charac- teristics of the unknown cultures against the descriptions of the Species in the literature. First, the characteris- tics of each isolated culture were checked individually against each Species listed in Bergey's Manual. When a cul- ture agreed generally with the description of a species listed in Bergey's Manual, the original reference to the species was found. This usually gave a more complete description. The second procedure followed in attempting to iden- tify the unknown cultures, was based on the fact that strains of a particular Species may vary markedly in their 43 antibiotic production and at the same time in their.morpho- logical characteristics. With this in mind it was thought wise to check the unknown cultures of this work against all Species listed in the literature as active antibiotic producers. There are several good publications in which attempts were.made to compile such a list. Of these, the largest number of antibiotics described and the species listed can be found in the article by Benedict (21) which lists 115 antibiotics. Approximately 89 Species account for all the antibiotics except those which are produced industrially by chemical.manipulation. The results of the cultural and morphological work are given in tables VI through X. At the end of each cul- ture's description are listed any known Species or species group to which the culture appeared to be related. Results and Discussion (No individual charts are found for organisms 58 and 415 or organisms 200, 201, 202, and 216. In the course of the work it became evident that organisms 218, 233, and 241 were three different species. Organisms 58 and 415 comprised a fourth Species, and organisms 200, 201, 202 and 216 composed a fifth species. Identification of the five species was attempted. wa- ever, after comparing their descriptions with the literature descriptions of no less than 150 species and strains, the 44 author found that none of the unknown organisms could be identified with a known species in more than a general way. This was due largely to the incomplete descriptions given for many organisms. In many cases reported in the literature, descriptions on three or four media were the basis for classifying a species (22). Three of the unknown species were placed into Specific groups with little or no indecision. There was little doubt that organism 218 belonged in the Streptomyces albus group. It agreed morphologically and culturally with the group characteristics. However, it resembled several of the species in this group without being unmistakably one or a variant of any of these species. For this reason the status of this organism could not be decided upon. Organism 200 belongs quite definitely to the §££§Ef tomyces flavus group. Its golden yellow pigmentation alone would seem.to warrent this. It resembles Streptomypes flaveolus and Streptomyces thermophglgg in several respects. Its identification is complicated not only by its entirely different set of characteristics depending upon the temp perature, but also by the fact that within the literature several species of the Streptomyces flavus group resemble each other so closely that with the Slight natural vari- ations they would be indistinguishable. An example of this may be found by comparing the descriptions of §tggp- 45 TABLE3VI DESCRIPTION OF ORGANISM 218 General Morphological Characteristics Spiral formation in aerial mycelium; long open spirals, spores are spherical or slightly oval observed on calcium.malate agar. Cultural Characteristics Observed on Various Media—at 35°C and Room Temperature Incubation 35°C Czapek's agar modified (glycerin) Growth colorless to cream Aerial white all over sur- mycelium face of growth Soluble none pigment Calcium.malate-glycerin agar Growth grayish tan; raised, leathery type growth Aerial white mycelium Soluble brown pigment Glucose aggr Growth no growth Aerial mycelium Soluble pigment Incubation Rbom Temperature same as 35°C same as 35°C none colorless to white vegetative white yellowish brown colorless changing to dark brown; raised, much wrinkled growth white changing to grayish black after 5 days dark brown to black 46 TABLEIVI continued Nutrient agar Growth colorless; flat & spreading Aerial tan, powdery surface mycelium Soluble none pigment Starch agar Growth tan, raised, spreading Aerial white, turning gray mycelium after 6 days Soluble none pigments Enzymatic none zone Potato dextrose agar Growth buff colored vegeta- tive, composing raised colonies;.much wrinkled & leathery Aerial none mycelium Soluble none pigment Gelatin Growth submerged, colorless; no ring formation Aerial none mycelium Soluble greenish brown pigment Liquefaction none same as 35°C same as 35°C none light gray vegetative; raised, smooth & spreading white, powdery with drop- lets on surface turning gray after 6 days none none smooth, round, raised colonies; light gray vegetative mycelium none none white growth none brown none Growth Aerial mycelium Soluble pigment H28 production Skim.milk Growth Coagulation Soluble pigment Litmus milk Growth Coagulation Reaction Reduction Peptoni- zation Growth 47 TABLEIVI continued I ' n Kligler s iron agar gray, raised, wrinkled growth; leathery & com- pact. none none yellowish brown ring formation; white aeri- al mycelium none greenish brown black ring formation; white aerial mycelium; brown soluble pigment none deeply alkaline none none Egapek‘s agar with sucrose none Temperature: same as 35°C none none reddish brown ring white aerial mycelium none slight brown yellowish brown ring; aerial.mycelium white; brown soluble pigment none alkaline none none none optimum 35°C 48 TABLE'VI continued Generic Name of Unknown Species and Probable Actinomyces Group to Which the Species Belongs Genus: Streptomyces (3. Pa929) Actinomyces group: Streptomyces albus group (22, pp. 28-29) 49 TABLE'VII DESCRIPTION OF ORGANISM 233 General merphological Characteristics Spiral formation in aerial mycelium; long, Open spirals; observed on calcium malate agar. Spores oval shaped. Cultural Characteristics Observed on Various Media at 35°C and Room Temperature Incubation 35°C Czapek's ggar modified (glycerin) Growth Aerial mycelium Soluble pigment Incubation Room Temperature colorless; scant, flat no growth and spreading white, scant none Calcium malate-glycerin agar Growth Aerial mycelium Soluble pigment Glucose agar Growth Aerial mycelium Soluble pigment yellowish brown; flat spreading tan to greenish tan green to greenish brown after 3 days cream Galored to dark brown; flat and spreading cream colored to greenish tan; powdery surface none white to dark yellow; flat & spreading white to tan changing to olive green, powdery surface none cream colored; flat, spreading white to tan with slight greenish tinge; powdery surface none Nutrient ggar Growth Aerial mycelium Soluble pigment Starch agar Growth Aerial mycelium Soluble pigment Enzymatic zone 50 TABLE VII continued flat, spreading; cream colored cream colored turning olive green; powdery none colorless changing to yellow after 6 days; flat & spreading white changing to tan after 6 days; powdery surface; concentric ring formation green, produced only after 14 days none to very slight Potato dextrose ggar Growth Aerial mycelium Soluble pigment Gelatin Growth colorless; flat & Spreading tan powdery surface with white powdery edges to the colony none light yellow ring for- mation; light yellow growth produced through out liquified portion same as 35°C same as 35°C none scant, flat; color- less changing to green- ish tan in 6 days white changing to greenish tan; powdery surface; concentric ring fo mation same as 35°C none buff colored; flat 8:: spreading white to pink with a slight greenish tinge to surface; powdery none same as 35°C 51 TABLEIVII continued Gelatin - continued Aerial white to light yellow mycelium Soluble none pigment Liquefaction 2" (total) in 14 days Kligler's iron agar Growth grayish brown; raised &.much wrinkled Aerial none mycelium Soluble none pigment H28 none production Skim.milk Growth light yellow ring formation Coagulation none Soluble none pigment Peptonization complete in 6 days Litmus milk Growth produced on surface of liquid; white aerial mycelium; light yellow vegetative Coagulation none Reaction Alkaline in 3 days Reduction none Peptonization 1 5/8" in 3 days same as 35°C none same as 35°C same as 35°C scant‘white none none light yellow ring formation none none 3/8" in 6 days same as 35°C none Alkaline in 5 days none 2" in 4 days; total (2") in 8 days. 52 TABLE}VII continued Czapek's agar with sucrose Growth none none Temperature: Optimum 35°C Generic Name of Unknown Species and Probable Actinomyces Group_to Which the Species Belongs Genus: Streptomyces (3, P. 929) Actinomyces group: Strgptomyces griseus gnbup (22, pp, 23-24; 25: PP. 14-16, 27-28; 26, pp. 259-269.) 53 TABLE VIII DESCRIPTION OF ORGANISM 241 General Morphological Characteristics Spiral formation in aerial mycelium; short compact spirals; observed on calcium.malate agar. Spores spherical in shape. Generally, some oval spores observed. Cultural Characteristics Observed on Various Aedia at 3500 and Rbom Temperature Incubation Incubation 3500 Rbom Temperature Czapek's ggar modified (glycerin) Growth flat; very slight no growth growth Aerial white, powdery, scant mycelium Soluble bright blue pigment Calcium malateeglycerin agar Growth colorless to white; no growth flat Aerial white turning gray mycelium in 5 days Soluble bright plum blue pigment Glucose agar Growth grayish black; gray growth;.much leathery growth, wrinkled ‘wrinkled Aerial none none mycelium Soluble deep violet none pigment Nutrient agar Growth Aerial mycelium Soluble pigment Starch agar Growth Aerial mycelium Soluble pigment Enzymatic zone 54 TABLE VIII continued scant, flat; no growth colorless white, powdery blue, rapidly Spreading light gray; flat no growth and Spreading buff colored; turning gray in 6 days bright blue 7 mm. Potato dextrose ggar Growth Aerial mycelium Soluble pigment Gelatin Growth Aerial mycelium Soluble pigment Lique- faction gray; smooth, raised, no growth colonial growth; entire edge; leathery none none submerged; very no growth slight growth none none complete (2") in 11 days 55 TABLEIVIII continued Kligler's iron ggar Growth grayish in color; small, round, smooth raised, much slightly raised colonial wrinkled growth; entire edge; gray in color Aerial white in color, scant none mycelium Soluble none none pigment H28 none none production Skim.milk Growth colorless, slight no growth Coagulation none Soluble reddish brown ring pigment suggests perhaps slight reddish pigment Litmus milk Genus: Growth reddish, brown ring no growth formation with cream colored aerial mycelium Coagulation none Reaction neutral Reduction none Peptoni- 3/8" in 8 days zation Growth Czapek's agar with sucrose Slight flat growth; scant, white aerial mycelium no growth Optimum 55°C Generic Name of Unknown Species Temperature: Streptomyces (3, p. 929) 56 TABLE IX DESCRIPTION OF ORG’NISMS 58 AND 415 General Morphological Characteristics Straight Sporulating hyphae, no Spirals on calcium.malate agar. Spores oval in shape. Cultural Characteristics Observed on Various Media at 35°C and Room Temperature Incubation 35°C Czapek's ggar modified (glycerigl Growth Aerial mycelium Soluble pigment light yellow vegeta- tive, flat surface growth in 3 days grayish white in color; covering the vegeta- tive growth completely in 3 days bright pink in 3 days Calcium malateiglycerine agar Growth Aerial mycelium Soluble pigment Glucqgg egg; Growth Aerial mycelium Soluble pigment yellow vegetative de- veloping deep into medium cream colored; thin rose or pink cream colored Spreading vegetative white, covering surface none Incubation Rbom Temperature white growth turning slight yellow in 3 days; scant. white; scant none colorless vegetative; mycelium turning yel- low in 5 days white, changing to greenish yellow in 5 days pink; very slight same as 35°C same as 35°C none Nutrient ggar Growth Aerial mycelium Soluble pigment Starch agar Growth Aerial mycelium Soluble pigment Enzymatic zone 57 TABLE31X continued flat, scant growth tan, powdery surface none light gray changing to brown after days white, changing to yellowish white in 8 days; powdery drop- lets on surface of medium none to very Slight after 13 days; slight pink in color 4 mm Potato dextrose ggar Growth Aerial mycelium Soluble pigment Gelatin Growth late in developing; grayish white vegeta- tive; smooth, raised, & flat colonies with diffuse edge. cream colored later becoming white to yellow white; powdery surface none yellow ring formation same as 35°C same as 35°C none same as 35°C yellowish white later developing slight greenish droplets on surface of medium none none cream colored vege- ‘tative; flat growth white going to light yellow; powdery surface none yellowish white ring formation just below surface of liquified portion after 4 days Aerial mycelium Soluble pigment 58 TABLE:IX continued Gelatin - continued yellowish white violet brown changing to rose after 14 days Liquifaction 2 inches in 15 days Growth Aerial mycelium Soluble pigment H28 production Skim milk Growth Coagulation Peptoni- zation Soluble pigment Litmus mnlk Growth Coagulation Change of reaction Reduction Peptonization Kligler's iron agar much wrinkled, raised; gray in color white covering entire surface none none yellowish white sur- face with white aerial mycelium none slight; throughout tube in 5 days; no zone formed none gray white surface none alkaline only after 12 days none slight after 16 days 1/16 inch. none light brown in liqui- fied portion only total in 16 days same as 35°C same as 35°C same as 350C none same as 35°C none 4" zone produced in 5 days none same as 35°C none same as 35°C none same as 3500 59 TABLE IX continued Czapek's ggar with sucrose Growth none none Temperature: Optimum 35°C Generic Name of unknown Species Genus: Streptomyces (3, P. 929) 60 TABLE X DESCRIPTION OF ORGANISKS 200, 201, 202, AND 216 ' General Morphological Characteristics Straight hyphae, no spirals noted at 35°C on either Czapek's agar (modified) or calcium-malate agar. Straight sporulating hyphae with some open spiraling noted at room temperature on Czapek's agar (modified) and calciumpmalate agar. oval in shape. Spores Cultural Characteristics Observed on Various Media at 35°C and Room Temperature Incubation at 35°C Czapek's ggar modified (glycerin) Growth Aerial mycelium Soluble pigment_ colorless to light yellow; flat & Spreading light yellow fluff with patches of white fluff none Ca1cium.malategg1ycerin agar Growth Aerial mycelium Soluble pigment Glucose agar Growth yellow; flat, spreading growth bright yellow powdery surface golden yellow tan to yellow brown; raised, much wrinkled surface Incubation Room Temperature Grayish white; flat and spreading gray; powdery with patches of white none gray gray aeriam with patches of white and yellow none deep tan to black vegetative; flat Spreading, and abundant Aerial mycelium Soluble pigment Nutrient agar Growth Aerial mycelium Soluble pigment Starch agar* Growth Aerial mycelium Soluble pigment Enzymatic zone Potato dextrose Growth Aerial mycelium Soluble pigment 61 TABLEIX continued Glucose agar - continued white powdery surface none colorless; flat, Spreading white powdery surface changing to tan with a pinkish tinge none golden yellow; flat and spreading sulfur yellow; powdery surface with white powdery patches none none agar* gray & golden vege- tative growth mixed together; flat & Spreading growth mouse gray powdery patches mixed with golden yellow powdery patches none mouse gray, powdery surface with patches of white fluff none white growth; flat, Spreading ash white covering entire surface of growth none gray yellow vegetative; flat & spreading growth gray and powdery with patches of white fluff none none same as 35°C gray powdery patches mixed with white patches which turn golden yellow after 8 days none Gelatin Growth Aerial mycelium Soluble pigment 62 TABLhtx continued yellow ring formation yellow ring formation; yellow colonies throughout tube none white none none Liquefaction total in 14 days (2") total in 14 days Kligler's iron agar Growth Aerial mycelium Soluble pigment ‘ S Sgoduction Skim milk Growth Coagulation Soluble pigment Peptoniza- tion Litmus milk Growth Coagulation tan; raised &.much grayish white; raised wrinkled &.much wrinkled none none none none none none yellow ring formation grayish.white ring formation none none none none Slight throughout tube %" zone formed at top after 6 days. No clear of tube after 6 days zoning colorless ring forma- same as 35°C tion; white aerial mycelium none none 63 TABLE:X continued Litmus milk - continued Reaction slightly alkaline alkaline after 7 days after 7 days Reduction none none Peptonization none 2" after 8 days Czapek's agar with sucrose Gnowth scant white powdery none surface growth; not penetrating into medium; no growth until after 14 days incubation Temperature: Optimum 35°C *The cultures of this Species varied in the amount of patching shown on certain media such as starch agar. For instance, sections of the cultures would be yellow while adjacent sec- tions would be gray, one color often predominating. By sub- culturing appropriate sections of any culture the predomina- ting colors could be changed. 7 Generic Name of Unknown Species and Probable Actinomyces Group to Which the Species Belongs_ Genus: Streptomyces (3, p. 929) Actinomyces group: Straptomyces flavus group (22, pp. 24-26; 24. PP. 457-465) 64 tomgces parvus and Streptomyaes cellulosaa (22)(23). Furthermore, it may possibly be that many different species of this group would be identical with each other if they were all described on the same set of media under the same conditions of pH and temperature. For these reasons, no conclusions were drawn as to the identification of or- ganism 200 with a particular species. Organism 233 was tentatively classified as a member of the Streptomyces griseus group. The greenish pigmenta- tion which is shown in the mycelium seems to justify this. This group is composed entirely of the variants of the species Straptomyces griseus which are separated on the basis of pigment and antibiotic production. The other two species studied do not fit into any known groups. Although they each have certain preperties which relate them to known species, it is the author's Opinion that neither has been reported previously. After comparing organism 241 to over 150 species and strains, only one known Species was found which had any major shmilarity to it. Organism 241 resembled Streptomyces coelicolor in its pigment production. Both pigments are litmus like in nature turning red in acid, and blue in alkali. Other than this characteristic, however, the two organisms are completely dissimilar both morphologically and culturally. Organism 241 is also definitely thermo- philic, while Straptomyces coelicolor is mesophilic. 65 By this comparison, it was concluded that organism 241 is not just a new strain of Streptomyces coelicolor, but in most probability a new Species. Organism 58 (or 415) is characterized by its rose colored soluble pigment formed on several media. Along with this pigment there is a brown colored one produced in gelatin. Because of these pigments, the organism can be compared with Streptomyces parpurascens and Streptomyces erythrochromqgenes, although it tends to resemble the for- mer more than the latter. In its vegetative growth and aerial mycelia, however, it tends to resemble Streptomyces eryth- rochromqgenes the closest and more so a Specific strain described by Waksman in 1919. Organism 58 is weak in its diastatic action on starch, and in this it resembles only Straptomyces erythrochromogenes (5)(22). By exhibiting weak proteolytic properties, however, it resembles only S3332- tomyces purpurascens (22)(27) of the two species. In Spite of these close similarities to both species, there seems to be enough significant differences shown in the comparisons of each to warrant a separation of organ- ism 58 from either. It was therefore concluded that or- ganism 58 is a different species but belonging to a hereto- fore uncharacterized group of organisms in which Strepto- myces pugpurascens and Streptomyces erythrochromogenes are also members. 66 The results obtained from this work on these five species verifies the conclusions reached by Routein and Finley, Benedict, Henrici, (loc cit.) and othervworkers in the field by further showing that the classification of unknown species of actinomycetes can be only tenta- tive until the work done in the past is better organized and a method of classification for the future is standardized. 67 5010mm A favorable set of conditions for isolating actinomy- cetes from an acid soil first had to be determined. Iso- lation of actinomycete cultures was carried out until as many culturally and morphologically different appearing cultures as possible were obtained. Twenty-eight cultures were finally chosen as representative of all types of ac- tinomycetes isolated. These 28 cultures were then screened for antibiotic activity against 12 bacterial species using the cross streak method. Penassay base agar gave the best results out of the four media used for screening. Twenty-three or 82.2% of the cultures were active against at least one of the bacterial species. A large number of the cultures were active against gram positive bacteria. Little activity was shown toward the gram negative bacteria except by eight actinomycete cultures which were actively antagonistic to both gram positive and gram negative bacteria. These eight highly active cultures, along with one inactive culture were characterized both morphologically and culturally in order to compare them with known actino- mycete Species. The nine cultures represented five dif- ferent species. Of these, three organisms were identified as members of recognized groups. One unknown Species be- longed to the Streptomyces albus group, another to the 68 Streptomyces flavus group and the third to the Streptomyces griseus group. The other two cultures were tentatively classified as species unreported in the literature. -‘ .1. 2. 4. 69 LITERATURE CITED Waksman, S. A. 1945 Microbial Antagonisms and Antibiotic Substances. The Commonwealth Fund, New York, N.Y. -Origina1 References for Work Cited from Above Source- Garre, C. 1887 Uber Antagonisten unter den Bak- terien. Centr. Bakt. Parasitenk., a, 312-313. Greig-Smith, R. 1917 Contributions to our know- ledge of soil fertility; the agricere and bacteri- ogoxigg of soil. Proc. Linn. Soc. N.S. Wales, 5a, 1 2"]. o Kriss, A. E. 1940 The lysozyme in actinomycetes. Microbiologia (U.S.S.R.), 2, 32-38. Waksman, S. A. 1950 The Actinomycetes. 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